Peroral ciprofloxacin therapy impairs the generation of a protective immune response in a mouse model for Salmonella enterica serovar Typhimurium diarrhea, while parenteral ceftriaxone therapy does not

Abstract

Nontyphoidal Salmonella (NTS) species cause self-limiting diarrhea and sometimes severe disease. Antibiotic treatment is considered only in severe cases and immune-compromised patients. The beneficial effects of antibiotic therapy and the consequences for adaptive immune responses are not well understood. We used a mouse model for Salmonella diarrhea to assess the effects of per os treatment with ciprofloxacin (15 mg/kg of body weight intragastrically 2 times/day, 5 days) or parenteral ceftriaxone (50 mg/kg intraperitoneally, 5 days), two common drugs used in human patients. The therapeutic and adverse effects were assessed with respect to generation of a protective adaptive immune response, fecal pathogen excretion, and the emergence of nonsymptomatic excreters. In the mouse model, both therapies reduced disease severity and reduced the level of fecal shedding. In line with clinical data, in most animals, a rebound of pathogen gut colonization/fecal shedding was observed 2 to 12 days after the end of the treatment. Yet, levels of pathogen shedding and frequency of appearance of nonsymptomatic excreters did not differ from those for untreated controls. Moreover, mice treated intraperitoneally with ceftriaxone developed an adaptive immunity protecting the mice from enteropathy in wild-type Salmonella enterica serovar Typhimurium challenge infections. In contrast, the mice treated intragastrically with ciprofloxacin were not protected. Thus, antibiotic treatment regimens can disrupt the adaptive immune response, but treatment regimens may be optimized in order to preserve the generation of protective immunity. It might be of interest to determine whether this also pertains to human patients. In this case, the mouse model might be a tool for further mechanistic studies.

Fig 1

Model system to study effects of antibiotic treatment of Salmonella infection. At day 0, streptomycin-treated mice were infected with S. Typhimurium^att (S. Tm^att; 5 × 10⁷ CFU intragastrically). At day 2 p.i., the mice were treated with either ciprofloxacin (15 mg/kg by gavage 2 times/day) or ceftriaxone (50 mg/kg i.p.) for 5 days. A first group of mice (group I) was sacrificed at day 5 p.i. to assess the impact of antibiotic treatment on gut inflammation and S. Typhimurium^att colonization of the gut. S. Typhimurium^att excretion was analyzed in groups II and III until day 40 p.i. At day 40 p.i., group II was sacrificed to determine intestinal inflammation and the presence of S. Typhimurium^att-specific antibody levels in serum and gut washes of immunized mice. Group III was pretreated with ampicillin (20 mg orally) and challenged with ampicillin-resistant wt S. Typhimurium Amp^r (200 CFU orally) at day 40 p.i. The degree of S. Typhimurium-induced gut inflammation was analyzed 2 days postchallenge.

Fig 2

Effects of ciprofloxacin treatment on the course of the primary diarrheal S. Typhimurium^att infection. (A) Time course of fecal S. Typhimurium^att shedding over a time period of 40 days. Streptomycin-treated mice were infected with S. Typhimurium^att. At day 2 p.i., S. Typhimurium^att-infected mice were treated with either PBS (control group; open circles) or ciprofloxacin (15 mg/kg by gavage 2 times/day; black circles) for 5 days, and S. Typhimurium shedding was monitored (5 < n < 23 individual mice per time point). (B) A group of mice (group I) was sacrificed at day 5 p.i., and cecum pathology was analyzed. (C) S. Typhimurium^att loads in cecum content (black circles), MLN (red circles), and the spleen (blue circles) at day 5 p.i. (D) A group of mice was sacrificed at day 40 p.i. (group II, in green). Cecum pathology at day 40 p.i. of control and ciprofloxacin-treated mice. (E) S. Typhimurium^att loads in cecum content (black), MLN (red), and the spleen (blue) at day 40 p.i. Dashed lines, detection limit for feces, cecum content, and MLN (10 CFU/g) and for spleen (20 CFU/g); black bars, median; **, P < 0.005; ***, P < 0.0005; ns, not significant.

Fig 3

Ciprofloxacin-treated mice display reduced protection in challenge infections. (A) S. Typhimurium^att-immunized mice had been treated with PBS (control mice; open circles) or ciprofloxacin (black circles). At day 40 p.i., the animals were challenged with wt S. Typhimurium (200 CFU intragastrically, group III). At day 2 postchallenge, inflammation of the cecal mucosa was assessed (a score of ≤3 indicates no inflammation; dashed line). (B) wt S. Typhimurium loads in the cecum content (black circles), MLN (red circles), and the spleen (blue circles). Dashed line, detection limits; black bars, median; *, P < 0.05; ns, not significant.

Fig 4

Effects of ceftriaxone therapy course of primary diarrheal S. Typhimurium^att infection. (A) Time course of fecal S. Typhimurium^att shedding. Streptomycin-treated mice were infected with S. Typhimurium^att. At day 2 p.i., the infected mice were treated with either PBS (control group; open triangles) or ceftriaxone (50 mg/kg i.p.; black triangles) for 5 days, and S. Typhimurium excretion was monitored over a period of 40 days (5 < n < 29 individual mice per time point). Dashed line, detection limit (≤10 CFU/g) for S. Typhimurium^att in feces. (B) At day 5 p.i., mice from the control and the ceftriaxone-treated groups were sacrificed (group I) and cecum pathology was analyzed. Dashed line, detection limit (a cecum pathology score of ≤3). (C) S. Typhimurium^att loads in cecum content (black triangles), MLN (red triangles), and the spleen (blue triangles) at day 5 p.i. (D) Cecum pathology of control and ceftriaxone-treated mice sacrificed at day 40 p.i. (group II). (E) S. Typhimurium^att loads in cecum content (black triangles), MLN (red triangles), and the spleen (blue triangles) at day 40 p.i. (group II). Black bars, median; *, P < 0.05; **, P < 0.005; ***, P < 0.0005; ns, not significant.

Fig 5

Ceftriaxone treatment does not interfere with the generation of a protective immune response. (A) Ceftriaxone-treated mice are protected from gut inflammation. Mice which had been exposed to S. Typhimurium^att and treated with PBS (control mice; open triangles) or ceftriaxone (black triangles) were analyzed in challenge infections. At day 39, animals were pretreated with ampicillin (20 mg orally) and challenged with wt S. Typhimurium (200 CFU orally) at day 40 p.i. Cecum inflammation was determined at 2 days postchallenge (a score of ≤3 indicates no inflammation; dashed line). (B) wt S. Typhimurium loads in the cecum content (black triangles), MLN (red triangles), and spleen (blue triangles). Dashed line, detection limit; black bars, median; ns, not significant.

Fig 6

S. Typhimurium-specific antibody levels present in ceftriaxone- and ciprofloxacin-treated animals. Antibody responses were analyzed in mice at days 5 and 40 p.i. with S. Typhimurium^att (groups I and II from Fig. 2 and 4). (A) Antibodies from serum or gut washes which were directed against the surface of S. Typhimurium^att were analyzed by bacterial FACS (2 < n < 10 samples per group; see Materials and Methods). The y axes indicate S. Typhimurium^att-specific lg levels (relative fluorescence units [rfu]), and the x axes indicate the respective dilutions of serum (1:20, 1:60, 1:180) or gut wash (1:2, 1:6, 1:18). (B) Western blot analysis. Serum and gut wash from control or ciprofloxacin- or ceftriaxone-treated mice (day 40 p.i.) were analyzed by immunoblotting against different bacterial lysates (E. coli, S. Enteritidis, S. Typhimurium^att, S. Typhimurium^att digested with proteinase K [PK]). Specific IgA or slgA was detected with an HRP-labeled secondary antibody. The results are representative of experiments with 2 different animals. (C) Antibody levels do not correlate with protection in ciprofloxacin-treated mice (n = 6). The cecum pathology score was plotted against the highest value (1:20 dilution for serum and 1:2 dilution for gut wash) of S. Typhimurium^att-specific IgG, IgA, and sIgA detected by bacterial FACS.